Project Summary Most organisms have an internal circadian timing system that organizes physiological and behavioral processes with respect to one another and the external environment to maximize organismal fitness. In modern society, many people live in conflict with the dictates of their internal clock, and this circadian dysregulation is associated with increased incidence of cancer, metabolic disease, mood disorders, cardiovascular disease and cognitive deficits. The circadian system is made up of central clock cells in the brain, input pathways that synchronize the clock to external environmental cues, and output pathways that couple the clock to overt physiological and behavioral processes. The output pathways are the least understood aspect of circadian rhythms; thus, identification of the cells and molecules that make up output pathways is of outstanding interest. To that end, we recently showed that the Drosophila pars intercerebralis (PI) is a major component of the circadian output pathway controlling rest:activity rhythms. In this proposal we will extend on those findings to further delineate the molecular and cellular mechanisms through which circadian information is transmitted across output pathways to control behavior. In Aim 1a, we will induce PI- specific knockdown of genes isolated through single-cell transcriptome analysis of PI cells to identify novel circadian output molecules. In preliminary experiments, we identified the slowpoke potassium channel as a regulator of rest:activity rhythms, and in Aim 1b, we will use ex vivo calcium imaging to test the hypothesis that slowpoke contributes to the transmission of circadian information by producing rhythms of PI neuron excitability. In Aims 2-3, we will investigate how a single central clock regulates multiple circadian outputs through the use of a newly-developed assay that for the first time allows for extended, uninterrupted analysis of feeding behavior. In Aim 2, we will induce genetic manipulations that excite, inhibit, or ablate specific molecularly-defined subsets of PI cells to test the hypothesis that control of feeding and rest:activity rhythms diverges at the level of the PI output cell. In Aim 3, we will perform genetic manipulations that selectively alter circadian clock speed in the brain or fat body, a peripheral clock tissue that is functionally equivalent to the mammalian liver and adipose, to investigate the relative contribution of central and peripheral clocks to behavioral outputs. Together, these experiments will identify genes that contribute to circadian outputs, map the action of these genes to specific cellular components of the output pathway, determine how the central clock controls distinct behavioral outputs, and assess how central and peripheral clocks coordinately modulate behavior. This will address several longstanding questions in the field and contribute to our understanding of the negative consequences of circadian disruption.